Synthesis and Characterization of Novel Fentanyl Derivatives

Fentanyl citrate, NLX, NLXM, guanosine 5′-[γ-thio]triphosphate tetralithium salt (cold GTPγS) and guanosine 5′-diphosphate sodium salt (GDP) were purchased from Sigma-Aldrich (Taufkirchen, Germany). [³H]-DAMGO and [³⁵S]-GTPγS were purchased from Perkin Elmer (Rodgau-Jügesheim, Germany). Isoflurane was purchased from AbbVie (Ludwigshafen, Germany), and CFA was purchased from Calbiochem (La Jolla, CA, USA).
FF6 (base) was synthesized by a contractor (ASCA GmbH, Berlin, Germany) (Fig. 1). The experimental measurement of pK_a was performed by a contractor (Sirius Analytical Ltd., Forest Row, UK). For in vitro experiments, fentanyl and FF6 were initially dissolved in water and dimethyl-sulfoxide (DMSO), respectively, and diluted in assay buffer to final concentrations. For in vivo experiments, FF6 was dissolved in DMSO and diluted with 0.9% NaCl to obtain the final concentrations. The maximum DMSO concentration was 4.2% for s.c., and 0.5% for i.v. injections. Fentanyl, NLX and NLXM were dissolved in water and diluted with 0.9% NaCl. Control groups were treated with vehicle (DMSO or NaCl, respectively). In the previously described fentanyl derivatives, fluorination of the ethylidene bridge yielded FF3 (experimental pK_a = 7.22)^{3 (link)}, and fluorination of the piperidine ring lead to the compound NFEPP (experimental pK_a = 6.82)^{15 (link)} (Fig. 1 and Table 1).